RESEARCH ARTICLE
Comparison of neurological health outcomes
between two adolescent cohorts exposed to
pesticides in Egypt
Ahmed A. Ismail1,2, Matthew R. Bonner3, Olfat Hendy4, Gaafar Abdel Rasoul2, Kai Wang5,
James R. Olson3,6, Diane S. Rohlman1,7*
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1 Department of Occupational and Environmental Health, College of Public Health, University of Iowa, Iowa
City, Iowa, United States of America, 2 Community Medicine and Public Health Department, Faculty of
Medicine, Menoufia University, Shebin El-Kom, Egypt, 3 Department of Epidemiology and Environmental
Health, State University of New York, Buffalo, New York, United States of America, 4 Department of Clinical
Pathology, National Liver Institute, Menoufia University, Shebin El-Kom, Egypt, 5 Department of Biostatistics,
College of Public Health, University of Iowa, Iowa City, Iowa, United States of America, 6 Department of
Pharmacology and Toxicology, State University of New York, Buffalo, New York, United States of America,
7 Oregon Institute for Occupational Health Sciences, Oregon Health & Science University, Portland, Oregon,
United States of America
* [email protected]
OPEN ACCESS
Citation: Ismail AA, Bonner MR, Hendy O, Abdel
Rasoul G, Wang K, Olson JR, et al. (2017)
Comparison of neurological health outcomes
between two adolescent cohorts exposed to
pesticides in Egypt. PLoS ONE 12(2): e0172696.
doi:10.1371/journal.pone.0172696
Editor: Pieter Spanoghe, Universiteit Gent,
BELGIUM
Received: August 18, 2016
Accepted: February 8, 2017
Published: February 23, 2017
Copyright: © 2017 Ismail et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data of
the study are contained in the uploaded supporting
information.
Funding: The work was supported by the Fogarty
International Center and the National Institute of
Environmental Health Sciences (R21 ES017223
and R01 ES022163).
Abstract
Pesticide-exposed adolescents may have a higher risk of neurotoxic effects because of
their developing brains and bodies. However, only a limited number of studies have
addressed this risk among adolescents. The aim of this study was to compare neurological
outcomes from two cohorts of Egyptian adolescents working as pesticide applicators. In
2005 and 2009, two cohorts of male adolescents working as pesticide applicators for the
cotton crop were recruited from Menoufia Governorate, Egypt. The same application schedule and pesticides were used at both times, including both organophosphorus, and pyrethroid compounds. Participants in both cohorts completed three neurobehavioral tests,
health and exposure questionnaires, and medical and neurological screening examinations.
In addition, blood samples were collected to measure butyryl cholinesterase (BChE) activity.
Pesticide applicators in both cohorts reported more neurological symptoms and signs than
non-applicators, particularly among participants in the 2005 cohort (OR ranged from 1.18 to
15.3). Except for one test (Trail Making B), there were no significant differences between
either applicators or non-applicators of both cohorts on the neurobehavioral outcome measures (p > 0.05). The 2005 cohort showed greater inhibition of serum BChE activity than the
2009 cohort (p < 0.05). In addition, participants with depressed BChE activity showed more
symptoms and signs than others without BChE depression (p < 0.05). Our study is the first
to examine the consistency of health outcomes associated with pesticide exposure across
two cohorts tested at different times from the same geographical region in rural Egypt. This
similar pattern of findings across the two cohorts provides strong evidence of the health
impact of exposure of adolescents to pesticides.
Competing interests: Oregon Health and Science
University and Dr. Rohlman have a significant
financial interest in Northwest Education Training
and Assessment, LLC, a company that may have a
PLOS ONE | DOI:10.1371/journal.pone.0172696 February 23, 2017
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Pesticide health effects
commercial interest in the results of this research
and technology. This potential conflict of interest
was reviewed by the University of Iowa and Oregon
Health and Science University and an approved
Conflict of Interest in research management plan
was implemented. This does not alter our
adherence to PLOS ONE policies on sharing data
and materials.
Abbreviations: ANOVA, Analysis of Variance;
BARS, Behavioral Assessment and Research
System; BChE, Butyryl cholinesterase; M-H OR,
Mantel-Haenszel common odds ratio (M-H OR);
OP, Organophosphorus; OR, Odds Ratio; WAIS-R,
Wechsler Adult Intelligence Scale—Revised
(WAIS-R).
1. Introduction
Adolescents are occupationally exposed to pesticides while performing a range of tasks during
the pesticide application process. These tasks range from applying with a backpack sprayer,
mixing and loading pesticides, cleaning the equipment or holding signs to mark the edges of
the field during the application process [1]. Pesticide application to the cotton crop in Egypt is
managed and regulated by the Egyptian Ministry of Agriculture. Pesticides are applied during
the summer at 4 time-points according to the life cycles of different cotton worms: cotton bollworms (pink and spiny) and cotton leafworms [2]. Early in the growing season, a biological
bacterial insecticide is applied as a growth promoter, followed by 3 cycles of pesticide application throughout July and August to control cotton worm infestations. The applied insecticides
include organophosphorus (primarily chlorpyrifos) and pyrethroid compounds [1].
Previous research examining long-term occupational exposure among adults, without previous poisoning, has demonstrated increased reporting of a wide range of neurological symptoms and signs, most commonly fatigue, headache, blurred vision, dizziness [3–5], but also
depression, difficulty with concentration or memory, irritability, and numbness [6, 7]. Others
have reported nerve function abnormalities, paresthesia, increased vibration sensitivity, balance difficulties, tremors, staggering and weakness, hyper-reflexia and loss of muscle strength
in legs or arms, and difficulty in moving fingers or grasping objects [4, 8, 9]. Neurobehavioral
performance was also examined among adult pesticide workers; it was found that workers
exposed to organophosphorus (OP) pesticides demonstrate deficits in response speed and
coordination, sustained attention, visual perception, and memory [10, 11]. More years working in agriculture and handling pesticides is associated with increased neurobehavioral deficits
[12, 13]. Few studies have addressed these outcomes among adolescent pesticide applicators
[1, 14–17]. The most commonly reported neurobehavioral deficits among adolescents were
motor speed and coordination, information processing speed and executive functioning,
attention, and memory [1, 14, 16]. Adolescents working in agriculture showed more neurological symptoms, e.g. headache, tremors, insomnia, dizziness, irritability, and numbness, than
adolescents not working in agriculture [15, 17].
Cholinesterase activity as a biomarker of effect is often used to characterize exposure to OP
pesticides, where the common mechanism of OP neurotoxicity is the inhibition β-cholinesterases: acetyl cholinesterase (AChE) and butyryl cholinesterase (BChE), with a more sensitivity
of BChE [18]. Several studies with adults have found that cholinesterase activity decreased
after exposure [19–21]. Other studies reported lower cholinesterase levels among exposed participants than control participants [11, 22]. Studies with adolescents have also demonstrated
lower cholinesterase activity among adolescent applicators compared to non-applicators
[1, 16, 18]. Although numerous studies have examined the relationship between neurological
symptoms reporting and cholinesterase activity [1, 4–7, 17, 21, 23], there is a scarcity of the
studies that interested in studying this relationship among adolescents.
Adolescents may be at greater risk from pesticide exposure than adults because of their
still developing bodies [24, 25]; Furthermore, they may perform agricultural tasks that put
them at risk for exposure, particularly in countries where there are few restrictions addressing
children’s work in agriculture [1, 19]. This demonstrates a need to examine the impact of
exposure to pesticides on neurological outcomes among adolescents occupationally working
in agriculture. In order to advance our understanding, it is important to look for converging
evidence, replicating the neurological and neurobehavioral findings of similar cohorts who are
exposed in the same way and evaluating them using the same tools is needed to confirm the
relationship between exposure of adolescents to pesticides and the deleterious effects of
pesticides.
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Pesticide health effects
Adverse effects associated with pesticide exposure were first identified in a cohort of adolescents in 2005 [1]. A second study in 2009 was conducted to determine if similar effects were
found and to evaluate additional outcome measures [16]. Similar application schedules and
pesticides were applied at both time points. The goal of the current work was to examine the
impact of pesticide exposure on health outcomes, including: neurobehavioral symptoms and
signs, neurobehavioral performance, and butyryl cholinesterase (BChE) activity among the
two cohorts of participants examined in 2005 and 2009, and evaluate whether these effects are
consistent or can be replicated over time.
2. Methods
2.1. Participants
Participants for both cohorts were recruited from Menoufia Governorate, and testing was carried out in August of 2005 and 2009, at the end of the pesticide application season. The season
begins in June and ends in August. During this time, approximately 10 adolescents from each
village were hired by the local stations of the Ministry of Agricultural to apply pesticides to the
cotton crop under supervision of adult agriculture engineers and employees. Adolescents
hired by the Ministry of Agriculture, between 12 and 18 years of age, were recruited in 2005
and 2009 (N = 41 in 2005 and N = 21 in 2009). There was a high response rate at both time
periods (89.3% in 2005 and 91.3% in 2009). Adolescents who had never worked in the cotton
fields was also recruited through friends and relatives of the applicator adolescents (N = 38 in
2005 and N = 20 in 2009) as a comparison group. Non-applicators lived in the same community as applicators and attended the same schools, but were not interested in working as pesticide applicators. The pesticide application process was the same at both years and is described
in detail elsewhere [1]. The protocol and consent forms used in this study were approved by
the Oregon Health & Science University (USA) and Menoufia University (Egypt) Institutional
Review Boards. Participants and their legal guardians gave written informed consent prior to
enrollment.
2.2. Procedure
Both cohorts, 2005 and 2009, completed questionnaires, provided a blood sample and performed a battery of neurobehavioral tests. The 2009 participants also provided a urine sample
for the measurement of 3,5,6-trichloro-2-pyridinol (TCPy); a specific metabolite for chlorpyrifos, the primary pesticide applied. Although a subset of tests was the same at both years, in
2005, all neurobehavioral tests were traditional tests from the Wechsler Adult Intelligence
Scale—Revised (WAIS-R). In 2009, a series of computerized tests from the Behavioral Assessment and Research System (BARS) replaced some of the individually administered tests
(Table 1). Data collection methods are described in brief below.
2.2.1. Questionnaires. For both cohorts, adolescents with the assistance of their parents,
completed a questionnaire describing their medical and work history, including information
about their exposure to pesticides. The questionnaire included items asking about the frequency of neurological symptoms e.g. headache, pain, and fatigue. In 2009, detailed questions
were added to the questionnaire to get a complete profile of exposure to pesticides at work,
home, and in family fields.
2.2.2. Medical examination. Detailed clinical medical examinations and complete neurological examinations were administered by specialists during both years. These examinations
included assessment of the following signs among study cohorts: tremors, and neurological
incoordination, in addition to any abnormalities in ankle and knee reflexes, superficial and
deep sensations, or in muscle power.
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Pesticide health effects
Table 1. Neurobehavioral functions assessed and the tests administered for both cohorts (2005 and 2009).
Functions
Tests
Memory
Match To Sample; MTS a
2005
p
Serial Digit Learning; SDL a
Benton Visual Retention; BVRT
Attention/Short memory
b
WAIS-R Digit Span; DS b
Digit Span Test; DST a
WAIS-R Arithmetic b
Sustained Attention
Information Processing Speed
Simple Reaction; SRT a
Visual Motor
WAIS-R Digit Symbol b
p
p
p
Symbol Digit; SDT
p
p
p
p
a
Trail Making A & B b
WAIS-R Block Design b
p
p
a
Finger Tapping; TAP
Perception
p
Continuous Performance; CPT a
Motor Speed/Coordination
2009
p
p
p
p
p
Format
Computer
Computer
Paper-pencil
Paper-pencil
Computer
Paper-pencil
Computer
Computer
Computer
Paper-pencil
Computer
Paper-pencil
Manual
doi:10.1371/journal.pone.0172696.t001
2.2.3. Neurobehavioral test battery. Age appropriate versions of tests from the Wechsler
Adult Intelligence Scale—Revised (WAIS-R; the current version available at the time) [26], validated in an Arabic-speaking population [27], were used to assess neurobehavioral function.
In addition, the 2009 cohort also completed computerized neurobehavioral tests from the
Behavioral Assessment and Research System (BARS; Table 1) [28]. All test instructions were
translated into Arabic. Examiners read the instructions to participants for non-computerized
tests, while for the computerized tests, instructions were presented in Arabic on the screen and
also simultaneously delivered orally through headphones. Reliability and validity of BARS tests
were approved for the Arabic speaking populations [16, 29]. For the purpose of the current
study, the comparison between both cohorts was done for the three neurobehavioral tests that
were administered identically to both cohorts.
2.2.4. Butyryl cholinesterase activity. Cholinesterase enzyme activity, as a biomarker of
effect chlorpyrifos, was evaluated at the end of the application season for both study cohorts.
The Weber method [30] was used to measure the serum cholinesterase enzyme (Butyryl cholinesterase; BChE) in 2005 through a standard laboratory kit (Test-combination Boehringer
Mannheim GmbH Diagnostica). In 2009, Butyryl cholinesterase activity was measured in
whole blood using the EQM Test-Mate kit (EQM Research, Cincinnati, OH, USA) [31], which
is based on the Ellman method [32].
2.2.5. Data analysis. SPSS version 23 was used for data analysis. Analysis of Variance
(ANOVA) test was employed to test the difference between cohorts (2005 and 2009), across
job categories (applicators and non-applicators), and the interaction between cohorts and job
categories. Odds ratios (ORs) and their confidence intervals were used to estimate the risk of
developing symptoms and signs between groups. These ORs for both cohorts were contrasted
to test their homogeneity using chi-square tests and p-values. When the results were homogenous, Mantel Haenszel common OR was used to test the significance of ORs over the two
study cohorts. Regression analysis was performed to examine the differences in performance
on the three neurobehavioral tests administered to both cohorts, between applicators and nonapplicators across the two cohorts. Differences of adjusted means and their standard errors are
presented for the neurobehavioral tests controlling for age and years of education. The differences in number of symptoms between cohorts, job categories, and also beween depressed and
non-depressed BChE activity participants were analyzed using the Mann-Whitney test. Chisquare test was used to examine the differences between cohorts (within job categories) and
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Pesticide health effects
Table 2. Comparison of demographic and exposure characteristics of applicators and non-applicators in 2005 (n = 79), and 2009 (n = 41).
Characteristics 2005
Non-applicators
(n = 38) Mean (SD)
2009
Applicators (n = 41)
Mean (SD)
Non-applicators
(n = 20) Mean (SD)
P-value
Applicators (n = 21)
Mean (SD)
Cohorta Job
categoryb
Interactionc
Age (y)
15.4 (1.7)
15.2 (1.7)
15.5 (1.5)
15.5 (2.1)
0.6
0.8
0.8
Education (y)
7.8 (3.8)
6.8 (4.2)
9.5 (1.5)
9.3 (1.5)
< 0.001
0.2
0.4
Days worked
22.4 (6.9)
18.9 (7.4)
0.08
Years worked
5.5 (2.3)
2.1 (1.0)
< 0.001
a,
comparison between 2005 and 2009 cohorts
b,
comparison between applicators and non-applicators across the two years of the study
Interaction between job category and cohort
c,
doi:10.1371/journal.pone.0172696.t002
between job categories (within cohorts) among the participants with depressed BChE activity.
Pearson correlation was run to test the correlation between neurobehavioral tests and BChE
activity for both cohorts.
3. Results
3.1. Characteristics of participants
Table 2 describes the demographic characteristics of participants in both the 2005 and 2009
cohorts. Tests compare the mean responses between cohorts (within job categories), between
job categories (within cohorts), and modification of differences between job categories over
time (interaction effect). Means and standard deviations (SD) were reported and compared
with ANOVA test. Age was not significantly different between all groups, averaging 15.4 years
overall. The 2009 participants had more years of education than the 2005 participants, but
there were no differences in years of education between applicators and non-applicators of
both cohorts. While the 2005 applicators worked significantly more years in agriculture than
the 2009 applicators (p < 0.001), the difference in days worked in pesticide application in 2005
and 2009 was not significantly different between applicators of both cohorts (p = 0.08).
3.2. Neurological manifestations
Table 3 shows that applicators in both cohorts reported higher frequencies of neurological
symptoms and signs than non-applicators, but only the 2005 applicators reported significantly
more neurological symptoms and signs than non-applicators (20 out of the 25 manifestations
had significant 95% CI). A test of homogeneity of ORs of both cohorts for each symptom was
conducted to see if the ORs of both cohorts were different. Outcomes of the homogeneity OR
test indicated that ORs of both cohorts for all symptoms are homogenous, p > 0.1 (Table 3,
the 2nd right column). In the last column of Table 3, the Mantel Haenszel common OR (M-H
OR) is used for all symptoms and signs to examine the significance of common OR across the
two cohorts. Common ORs are significant for most of the neurological symptoms and signs;
this indicates higher frequencies of symptoms and signs among applicators than non-applicators. All reported ORs were unadjusted due the small sample sizes of both cohorts. The median
number of symptoms and signs was also significantly higher among applicators than nonapplicators (within cohorts, p = 0.03) and higher for the 2005 cohort than that of the 2009
cohort (p = 0.005) (Data not shown).
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Pesticide health effects
Table 3. Comparison of the neurological symptoms and signs between job categories in each cohort (OR and CI), and homogeneity evaluation of
the comparisons across the two cohorts (X2, p-value) and the significance of the Mantel-Haenszel common odds ratio (M-H OR (95% CI).
Signs/Symptoms 2005
X2 (1df), p M-H OR (95% CI)
2009
Nonapplicators
(n = 38) N (%)
Applicators
(n = 41) N (%)
OR (95% CI)
Headache
9 (23.7)
15 (36.6)
Arthralgia
9 (23.7)
11 (26.8)
Nonapplicators
(n = 20) N (%)
Applicators
(n = 21) N (%)
OR (95% CI)
1.86 (0.70, 4.96) 2 (10.0)
4 (19.0)
2.12 (0.34, 13.1) 0.02, 0.90 1.92 (0.81, 4.54)
1.18 (0.43, 3.27) 2 (10.0)
3 (14.3)
1.50 (0.22, 10.1) 0.047,
0.83
1.25 (0.51, 3.06)
Pain
6 (15.8)
8 (19.5)
1.29 (0.40, 4.14) 2 (10.0)
2 (9.5)
0.95 (0.12, 7.46) 0.07, 0.79 1.20 (0.44, 3.30)
Fatigue
3 (7.9)
15 (36.6)
6.73 (1.76, 25.7) 1 (5.0)
4 (19.0)
4.47 (0.45, 44.0) 0.09, 0.76 6.06 (1.91, 19.2)*
Blurred vision
2 (5.3)
14 (34.1)
9.33 (1.95, 44.6) 1 (5.0)
3 (14.3)
3.17 (0.30, 33.3) 0.59, 0.44 6.92 (1.92, 25.0)*
Feeling
depressed
2 (5.3)
13 (31.7)
8.36 (1.74, 44.1) 1 (5.0)
4 (19.0)
4.47 (0.45, 44.0) 0.20,
0.66)
Difficulty in
concentration
2 (5.3)
12 (29.3)
7.45 (1.54, 36.0) 0
1 (4.8)
6.92 (1.91, 25.1)*
0.13, 0.72 8.11 (1.69, 39.0)*
Dizziness
2 (5.3)
12 (29.3)
7.45 (1.54, 36.0) 0
0
Difficulty in
understanding a
2 (5.3)
11 (26.8)
6.6 (1.36, 32.1)
1 (5.0)
2 (9.5)
2.00 (0.17, 24.0) 0.67, 0.41 4.86 (1.31, 18.0)*
Troubles in
remembering b
1 (2.6)
12 (29.3)
15.3 (1.88, 125)
1 (5.0)
4 (19.0)
4.47 (0.45, 44.0) 0.65, 0.42 9.56 (2.11, 43.4)*
Feeling irritable
1 (2.6)
11 (26.8)
13.6 (1.66, 111)
1 (5.0)
4 (19.0)
4.47 (0.45, 44.0) 0.52, 0.47 8.82 (1.93, 40.3)*
Numbness
1 (2.6)
9 (22.0)
10.4 (1.25, 86.6) 0
3 (14.3)
0.31, 0.58 14.0 (1.73, 114)*
Superficial
sensation
abnormality
2 (5.3)
12 (29.3)
7.45 (1.54, 36.0) 0
3 (14.3)
0.43, 0.51 9.44 (2.00, 44.6)*
Knee reflex
abnormality
2 (5.3)
10 (24.4)
5.81 (1.18, 28.5) 0
1 (4.8)
0.17, 0.68 6.43 (1.32, 31.3)*
Tremors
2 (5.3)
8 (19.5)
4.36 (0.86, 22.0) 0
1 (4.8)
0.22, 0.64 4.95 (1.00, 24.5)*
Incoordination
1 (2.6)
10 (24.4)
11.9 (1.45, 98.5) 2 (10.0)
4 (19.0)
Ankle reflex
abnormality
1 (2.6)
6 (14.6)
6.34 (0.73, 55.4) 0
1 (4.8)
0.15, 0.70 7.44 (0.87, 63.6)
Muscle power
abnormality
1 (2.6)
5 (12.2)
5.14 (0.57, 46.2) 0
1 (4.8)
0.19, 0.66 6.21 (0.71, 54.0)
Deep sensation
abnormality
0
1 (2.4)
a,
b,
0
2.12 (0.43, 13.1) 1.63, 0.20 5.27 (1.43, 19.4)*
0
Difficulty in understanding meanings of newspaper and books
Troubles in remembering things observed by relatives
doi:10.1371/journal.pone.0172696.t003
3.3. Neurobehavioral performance
There were no significant differences between applicators of both study cohorts and also no
significant differences for non-applicators on the neurobehavioral tests after controlling for
age and years of education, except that non-applicators in 2009 took a significantly longer time
to complete the Trail Making-B test than non-applicators in 2005 (Table 4).
3.4. Butyryl cholinesterase activity of both cohorts of the study
The number of participants with BChE inhibition were compared across the study cohorts;
however, different methods were employed to evaluate BChE activity in both cohorts. The
range of normal BChE activity reported by kit manufacturers (3.33–7.03 IU/ml for the 2005
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Pesticide health effects
Table 4. Differences in neurobehavioral performance between applicators and non-applicators of both cohorts.
Neurobehavioral Test
Non-applicators Difference (95% CI)
Applicators Difference (95% CI)
Block Design
3.9 (-1.5, 9.3)
4.1 (-0.02, 8.2)
Trail Making—A a
- 9.9 (-20.0, 0.3)
- 8.9 (-20.1, 2.2)
- 21.8 (-42.0, -1.7)*
- 8.7 (-24.5, 7.2)
Trail Making—B
a
a,
Differences represents (2005–2009) time in seconds. These are time measures tests, so, higher is worse
* p < 0.05
doi:10.1371/journal.pone.0172696.t004
cohort [30], and 1.35–3.23 U/ml for the 2009 cohort [31]) were used to identify participants
with depressed or low BChE activity relative to the respective normal range. For both cohorts,
a greater percentage of applicators had depressed levels of cholinesterase, relative to the normal
range, than non-applicators (Fig 1; 2005: chi-square = 7.9, p = 0.005; 2009: chi-square = 5.2,
p = 0.02).
Fig 1. Percent of participants with depressed BChE activity relative to the normal ranges in 2005 and 2009.
doi:10.1371/journal.pone.0172696.g001
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Pesticide health effects
Table 5. Median and interquantile range (IQR) of neurological symptoms and signs of the two cohorts according to BChE activity (in each cohort,
depression in BChE activity was determined relative to normal ranges from the methods used).
2005 (n = 79)
Median
IQR
2009 (n = 41)
Total (n = 120)
Depressed BChE
(n = 22)
Normal BChE
(n = 57)
Depressed BChE
(n = 8)
Normal BChE
(n = 33)
Depressed BChE
(n = 30)
Normal BChE
(n = 90)
4.0
0.0
0.0
0.0
1.5
0.0
10.2
2.0
2.2
1.0
9.2
1.0
p-value
0.006
0.75
0.013
a
a,
P-value of Mann-Whitney test
doi:10.1371/journal.pone.0172696.t005
3.5. Relationship between neurological symptoms and signs and butyryl
cholinesterase activity
Among participants in the 2005 cohort, participants with depressed BChE activity reported a
greater number of neurological symptoms and signs relative to participants that were within
the normal range of BChE activity (p = 0.006). This relationship was also observed when participants from the 2005 and 2009 cohorts were combined (p = 0.013). However, this difference
between participants with depressed and normal BChE was not found for the 2009 cohort
(p = 0.75) (Table 5).
3.6. Correlation between neurobehavioral performance and butyryl
cholinesterase
Pearson correlation was used to examine the correlation between neurobehavioral tests and
BChE activity for both study cohorts. A non-significant correlation between neurobehavioral
outcomes in both study cohorts and BChE levels was found, except for Trail making A and B
in the 2005 cohort. However, a significant negative correlation was present between the BChE
level and Trail Making A (r = -0.29, p = 0.01), and also with Trail Making B (r = -0.42,
p < 0.001) (Fig 2). This indicates impaired performance in these tests with more depression of
BChE activity.
4. Discussion
Our study is the first to examine the consistency of health outcomes associated with pesticide
exposure across two cohorts tested at different times from the same geographical region in
rural Egypt. Pesticide applicators in both cohorts reported more neurological manifestations,
neurobehavioral deficits, and BChE inhibition than non-applicators. The consistency of the
findings over the two cohorts of the study provides further evidence of the neurological health
effects of prolonged exposure to organophosphorus pesticides. The two cohorts were examined in 2005 and 2009 using a similar methodology with similar questionnaires, neurobehavioral testing, medical examination, and evaluation of BChE activity. During the two years of
the study, applicators also applied the same types of pesticides, and followed the same schedule
and methods of pesticide application. However, the 2005 cohort showed more neurological
symptoms and signs, and neurobehavioral deficits, which in part may be due to more years of
working in pesticide application and/or more extensive exposures, and fewer years of
education.
Pesticide applicators from both cohorts reported more neurological symptoms and showed
more signs than non-applicators of both study cohorts. Nevertheless, the significant ORs were
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Fig 2. Correlation between Trail making test (A & B) and butyryl cholinesterase (BChE) levels in participants from the
2005 cohort.
doi:10.1371/journal.pone.0172696.g002
obtained only among the 2005 cohort; the consistent higher frequencies of symptoms and
signs among applicators were confirmed through the homogeneity of OR as shown by the chisquare test, and the significant common OR by the Mantel-Haenszel test (Table 3). The results
of neurological signs and symptoms among adolescent pesticide applicators of both 2005 and
2009 cohorts agree with the findings of Egyptian studies that examined health hazards among
adult pesticide applicators [3, 11], and applicators and farmworkers worldwide: licensed private applicators [6], Florida farmworkers [10], and farm residents [7] in the USA; irrigation
workers in Ghana [33], Indonesian farmers [34], Sri Lankan farmers [4], Spanish greenhouse
sprayers [35], Indian pesticide manufacturing workers [22], and Emirates farmworkers [36].
This consistency of neurobehavioral findings is also found through comparison of neurobehavioral tests applied identically to both cohorts: Block Design, Trail Making A and B, where
applicators and non-applicators of both cohorts did not show any statistically significant difference except on the Trail Making B test (Table 4). Detailed comparisons of neurobehavioral
performance of applicators and non-applicators for each cohort are presented in other publications [1, 16]. The outcomes of the current study confirm the findings of other studies which
showed that functional domains most consistently affected by OP exposure include psychomotor and cognitive behavior [37]. These findings also were confirmed by a meta-analysis examining neurobehavioral performance among farmworkers and pesticide applicators [38].
Although pesticide applicators in 2009 did not show significantly more neurological symptoms and signs than the non-applicators, most likely due to the small sample size, this did not
affect the homogeneity of OR measured by the chi-square test or the significant common OR
by the Mantel-Haenszel test for the majority of the neurological symptoms and signs (Table 3).
These findings indicate an increase in frequency of both neurological symptoms and signs
among applicators compared to non-applicators, regardless of the examined cohort. The same
results were obtained when comparing the median of the number of symptoms and signs
experienced by each participant between applicators and non-applicators in each cohort and
across the two cohorts (last row-Table 3). This association between neurobehavioral deficits
and occupational exposure to OP pesticides among applicators of both cohorts is confirmed
by the findings of non-significant differences between 2005 and 2009 cohorts in age and days
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Pesticide health effects
worked in applying pesticides (Table 2), and also after controlling for both age and years of
education in the regression model for neurobehavioral outcomes (Table 4). The relationship
between OP exposure and neurobehavioral deficits is also strengthened because applicators
from both cohorts used the same chemical compounds, the same method of pesticide application and almost the same work duration in both seasons.
In evaluating the biological effects of pesticide exposure of the two cohorts, results found
that a greater percentages of applicators had depressed levels of BChE than non-applicators
(Fig 1). This is consistent with the findings of other studies [19–21]. While the relationship
between neurological symptoms and BChE depression is obvious (Table 5), this is not the case
regarding neurobehavioral performance, where only one test (Trail Making) showed a negative correlation with BChE levels in the 2005 cohort. This is similar to what was found among
adolescent pesticide applicators in Egypt, where only Information, Digit Span and Trail Making were correlated with BChE activity [1]. These results also agree with findings from RoldanTapia and colleagues (2005), that BChE activity is not a valuable tool to explain neurological
deficits among workers occupationally exposed to OP. Changes in BChE activity may be
attributed to several factors e.g. inter- or intra-individual or seasonal variability or factors such
as alcohol consumption [13].
The study was limited by the small sample size and only a single measurement of cholinesterase activity, which does not provide information about the inhibition of cholinesterase
activity during the application season. Although other biomarkers of exposure are available to
characterize exposure, e.g., TCPy, this information was only available for participants tested in
2009 [16]. Additional work examining the changes across the season is needed to understand
the impact of exposure on neurological outcomes and to estimating the dose response relationship between pesticide exposure and neurobehavioral outcomes.
In conclusion, replicating the health findings associated with pesticide exposure among
adolescent pesticide applicators tested in 2005 and 2009 in the Menoufia Governorate, Egypt
demonstrates the neurological drawbacks and neurobehavioral deficits among adolescents
occupationally exposed to OP pesticides. Neurological symptoms and signs were significantly
higher among applicators than non-applicators, especially for the 2005 cohort. Fewer days and
years worked in 2009 and also the small sample sizes may be the reasons that fewer neurological deficits were observed in 2009. These outcomes provide more evidence of the higher risk
the adolescents may be exposed to when they work with dangerous chemicals such as pesticides. Due to the large number of children working in agriculture around the world, including
those working on family farms [39], it is important to understand the impact of exposure on
health outcome in order to change policy.
Supporting information
S1 File. Data file. Replication_Data_Block_Design101011.sav”. This is the raw data file.
(SAV)
S2 File. Study questionnaire. EGAD Applicator Baseline Questionnaire.docx This is the questionnaire used for the current study.
(DOCX)
Acknowledgments
We thank the Egyptian Ministry of Agriculture and the adolescents for their participation. In
addition, we would like to thank Michael Laserev and Kristin Mara for their assistance in statistical analysis of the manuscript data. Finally, we would like to thank Mahmoud Ismail,
PLOS ONE | DOI:10.1371/journal.pone.0172696 February 23, 2017
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Pesticide health effects
Tameem Abou Eleinin and Mohammed Fouad and other members of the Research Team at
Menoufia University for their assistance with data collection. Part of this work was presented
in the 2010 American Public Health Association Annual Meeting, Denver, Colorado.
Author Contributions
Conceptualization: AI MB JO DR.
Data curation: AI KW DR.
Formal analysis: AI KW.
Funding acquisition: DR.
Investigation: AI MB OH GA JO DR.
Methodology: AI MB JO DR.
Project administration: AI GA DR.
Resources: JO OH GA DR.
Supervision: AI MB OH GA JO DR.
Validation: AI MB JO DR.
Visualization: AI MB JO DR.
Writing – original draft: AI DR.
Writing – review & editing: AI MB JO DR.
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